DESCRIPTION: Infective endocarditis is a serious illness that requires lengthy medical or surgical treatment, causes severe complications including congestive heart failure and stroke, and is often fatal, even when treated. The viridans streptococci as a group and Streptococcus sanguis in particular are a leading cause of this disease. Identification of streptococcal virulence determinants would lead to a better understanding of the pathogenesis of streptococcal endocarditis and to the identification of targets for prevention and therapy. We have applied signature-tagged mutagenesis (STM) to identify Streptococcus sanguis virulence factors for endocarditis. This approach relies on the infection process in an animal model to identify bacterial genes needed for disease causation. Our initial three-year project resulted in adaptation of an in vitro transposition STM technique for use with S. sanguis and the creation and screening of 800 tagged mutants. The current application proposes to use STM and non-STM approaches to examine the contribution of cell wall-anchored proteins and lipoproteins to endocarditis virulence. The S. sanguis strain being used for these analyses is also being sequenced by us and our collaborators and is near completion. The availability of the sequence will allow for the remainder of the mutagenesis to be performed using a targeted approach involving in vitro transposition into defined amplicons. This approach will be technically simpler than our previous STM analysis and will also be more informative, allowing for categorization of these genes as essential for normal growth in culture, essential for endocarditis virulence, or dispensable for both traits. This approach will provide a comprehensive analysis of the role of two classes of surface proteins in endocarditis virulence. We also expect the study to examine virtually all potential vaccine candidates in S. sanguis and to identify those that are most promising.